Cellular aging or cellular senescence is a universal attribute of normal non-Transformed cells that is manifested by morphological changes accompanied by an age-dependent loss of proliferative potential, including the failure of the cells to respond to exogenous growth factors. A variety of theories have been proposed to explain the phenomenon of cellular senescence. Experimental evidence suggests that the age-dependent loss of proliferative potential may be the function of a genetic program (see, e.g., Smith et al. (1980) Mech. Age. Dev. 13:387; and Kirkwood et al. (1975) Theor. Biol. 53:481). This evidence includes cell fusion studies with human fibroblasts in vitro that demonstrate that the quiescent cellular senescent phenotype is dominant over the proliferative phenotype (see, e.g., Pereira-Smith et al. (1982) Somatic Cell Genet. 8:731; and Norwood et al. (1974) Proc. Natl. Acad. Sci. USA 1:223) and that protein synthesis in senescent cells, prior to fusion with young cells, is required for the inhibition of DNA synthesis within the young nucleus of the heterodikaryon (see, e.g., Burmer et al. (1983) Exp. Cell Res. 145:708; and Drescher-Lincoln et al. (1984) Exp. Cell Res. 153:208). Also, microinjection of senescent fibroblast mRNA into young fibroblasts inhibits the ability of the young cell to synthesize DNA (see,e.g., Lumpkin et al. (1986) Science 232:393) and entry of the young cell into the S phase of the cell cycle (Lumpkin et al. (1985) Exp. Cell Res. 160:544). Further, unique mRNA species are amplified in senescent fibroblasts in vitro (see, e.g., Wellinger et al. (1986) J. Cell Biol, 34:203; Flemming et al. (1988) Proc. Natl. Acad. Sci. USA 85:4099; West et al. (1989) Exp. Cell Res. 184:138; and Giordano (1989) Exp. Cell Res. 185:399). It has also been suggested that an altered genetic program exists in senescent human fibroblasts, which involves the repression of c-fos expression at the transcriptional level (see, e.g., Seshadri et al. (1990) Science 247:205). Thus, there appear to be genotypic, as well as phenotypic differences between young and old cells.
The relationship between cellular aging in vitro and cellular aging in vivo
Many of the morphological, physiological and biochemical characteristics that distinguish young from old cells in living mammals are exhibited by such cells when they are cultured in vitro. For example, fibroblasts of the dermal layer of skin and the connective tissue layer underlying the epithelia of the inner wall of the gastrointestinal tract display characteristics of aging similar to those found for fibroblasts in culture. Also, cells in vivo share many characteristics of cultured fibroblasts, including similar biochemical characteristics and tight control of growth, so that characteristics that distinguish young from old cells in culture are applicable to cells in living mammals.
A number of attributes have been identified that differentiate young and aged cells. In culture, young cells exhibit higher responsiveness to growth factors and higher rates of DNA and protein synthesis than old cells. Young mammalian, including human, fibroblast cells in tissue culture appear healthy and clean; possess a regular, long, thin spindle-shaped morphology; are tightly packed in arrays on becoming confluent on culture substrata; do not overgrow one another; seldom have other than one nucleus; and produce little debris in the culture medium. Fibroblast cells that are old display many age-related characteristics, including flattened and irregular morphology, abnormally large size, sparse growth, low cell yield per unit area of culture substratum, a significant frequency of polynucleated cells, difficulty in trypsinization, the inability to grow to confluence and a high rate of production of debris in the culture medium. The morphological characteristics that distinguish young from old cells, as well as the high level of autofluorescence found in old cells, reflect the various physiological and biochemical characteristics that distinguish young from aged cells.
The proliferative potential of normal human diploid cells is finite and a function of the number of cumulative population doublings (see, e.g., Hayflick et al.. (1961) Exp. Cell Res. 25:585; and Hayflick (1985) Exp. Cell Res. 37:614). The lifetime of human cells under controlled conditions in vitro is reproducible, maximally about 45 to 60 cumulative population doublings for a primary culture, and is inversely proportional to the in vivo age of the donor from whom the cells were obtained (see, e.g., Martin et al, (1979) Lab. Invest. 23:86; Goldstein et al. (1969) Proc. Natl. Acad. Sci. USA 64:155; Schneider et al. (1976) Proc. Natl. Acad. Sci. USA 73:3584; and LeGuilty, et al. (1973) Gereontologia 19:303).
A measure of the "age" of fibroblasts in tissues of a living mammal, which normally divide periodically under tight growth control, is the number of cell divisions that have occurred between the cells and their predecessors at about the time of birth of the mammal. In cultures of normal fibroblasts, fibroblasts that have not been immortalized or transformed to a cancerous state, total proliferative capacity decreases as a function of the number of doublings that separates a culture from the primary culture from which it was derived. Because cell division occurs periodically and growth rate decreases rapidly as the finite life span of the cells is approached, there is typically a correlation between chronological age of the mammal and the age of particular cells measured by cell divisions between the cells and their predecessors at birth. The proliferative capacities of cells, such as fibroblasts in primary cultures established with normal dermal cells, are inversely related to donor age. Thus, fibroblasts in primary cultures established with normal cells from human adult dermis have a lower total proliferative capacity than fibroblasts in primary cultures established with normal cells from human fetal or newborn dermis.
The narrowly defined (within a few doublings) total proliferative capacity of normal fibroblasts, particularly of human origin, in culture is a manifestation of the tight proliferative control of such cells. This narrowly defined total proliferative capacity makes it possible to specify the total proliferative capacity of cells in vivo, such as fibroblasts of the dermis of the skin or the connective tissue layer underlying the intestinal epithelium, in living mammals in terms of a fixed number of doublings, limited to a narrow range characteristic of the species of the mammal (e.g., 45-60 for human fibroblast cells about 10 for murine fibroblast cells).
Under certain conditions cells that normally have a limited total proliferative capacity can be transformed to lose this limitation. Cultures of such cells can be passaged repeatedly without any apparent limit on number of passages; such cells are said to be immortalized. Immortalization is also a characteristic of cancer cells that, in addition to exhibiting little or no proliferative control, do not exhibit contact inhibition. The loss of limited total proliferative capacity characteristic of cells in culture that are transformed to the immortalized or cancerous state is often accompanied by an abnormally increased rate of proliferation or growth rate. Similarly, the loss of tight proliferative control that characterizes cancer cells in vivo is often accompanied by an abnormally increased growth rate of the cells.
Treatments that are designed to preserve the "young" phenotype of aging cells in vivo also invariably increase the growth rate of the treated cells. Treatments that artificially increase the rate of cell division or the total proliferative capacity of cells in culture beyond normal limits have been found to increase the risk of transformation to the immortalized or cancerous state. The risk of causing transformation by tampering with the tight proliferative control on cells, by tampering with growth rates and/or limits on total proliferative capacity, are presumably due to effects of such tampering on the gene expression. Further, treatments that increase growth rate or total proliferative capacity, such as epidermal and platelet-derived growth factors, insulin, glucocorticoids, extracts of Panax ginseng, and gibberellin plant hormones, are generally effective in preserving the young phenotype of only young cells, for which the treatments are needed least. Old cells that have proliferated at least about 80-90% of their normal life spans, respond little, if at all, to treatment with even much higher concentrations of these substances than those effective with younger cells.
Cellular aging in vivo
Skin, which is the external covering of the body, has two components: the epidermis that contains four layers and the dermis, also referred to as the corium, cutis, derma, or true skin, that contains a superficial papillary layer and a deep reticular layer. Collagen constitutes about 80% of the dry weight of the dermis and is the major fibrillar component of human skin. The dermis is composed of connective tissue that contains lymphatics, nerves and nerve endings, blood vessels, sebaceous and sweat glands, and elastic fibers that provide the elastic properties of the skin. The mature fiber contains about 90% elastin and two glycosaminoglycans are present at concentrations of about 2% and 0.1%, respectively (see, e.g., Braverman(1982) J. Invest. Dermatol. 78:434-443). The coarse branching fibers are entwined with collagenous fiber bundles in the reticular dermis. The fibers rise from the deeper layers of the papillary layer of the dermis and, as they rise towards the epidermis, they split repeatedly become finer and form a network.
As humans age, there are changes in the quantity and integrity of dermal elastic tissue (Warren et al. (1991) J. of the American Acad. Dermatology 25:751-760). Gross alteration in elastin leads to alterations in the appearance of the skin (see, Bryce et al. (1988) J. Invest. Dermatology 91:175-180; Kornberg et al. (1985) New Engl. J. Med. 312:771-774; Shelley et al. (1977) Br. J. Dermatol. 7:441-445). The association of changes in the fibers and the appearance of wrinkles indicates a causal relationship between the integrity of the elastic fiber network and the mechanical properties of the skin. Aging skin is characterized by initial elastogenesis followed by a slow spontaneous progressive degradation of the elastic fibers that leads to laxity and wrinkling. Studies of skin from subjects of various ages indicate that the degradation of elastic fibers that begins about age 30 and becomes marked after age 70 is a major feature of aging skin.
In understanding the process of aging of human skin, it is pertinent to understand the role of fibroblasts, particularly in the dermis of human skin and in the corresponding connective tissue layer underlying the integuments of other mammals and the epithelia of the inner wall of the gastrointestinal tracts of humans and other animals. Fibroblasts synthesize components that are required for maintenance of the structural, functional and cosmetic integrity of the skin and the structural and functional integrity of other surface tissues covered by epithelia. These components include collagen and elastin, which are fibrous proteins responsible for the three-dimensional architecture of skin and the other surface tissues, fibronectin, which is a protein responsible for cell anchorage and maintenance of cell morphology; and a number of proteinaceous growth factors essential for the maintenance of epithelia and basal cell layers and connective tissue layers underlying them. Available evidence indicates that protein biosynthetic activity of fibroblasts decreases significantly with age. For example, the rate of collagen synthesis at about 70% of life expectancy for fetal-derived human fibroblasts in culture is only about 50% of that of such fibroblasts at less than 20% of life expectancy.
The changes in the appearance of the skin with age result from natural or intrinsic aging superimposed by actinic damage resulting from photoaging (see, e.g., Weiss et al. (1988) J. American Medical Assoc. 259:527-532). Intrinsic aging includes changes that occur as a result of endogenous factors and genetically programmed senescence, including epidermal and dermal atrophy. Photoaging results from long-term exposure to UV (ultra-violet) radiation, primarily from the sun. UV exposure is also associated with tumor induction and other skin pathologies. UV radiation from the sun includes UVB (280-315 nm) and the more penetrating UVA (315-400 nm) radiation. UVB causes erythema, skin cancer and dermal connective tissue damage. UVA also causes erythema and is carcinogenic at higher doses. Low doses (2.5 minimal erythemic doses (MEDs)) are sufficient to cause endothelial cell enlargement, extravasation of blood cells, and perivenular neutrophil infiltrates as wells increased concentrations of mediators of the inflammatory response (see, e.g., Kligman et al. (1985) J. Invest. Dermatol. 84:272-276).
Long-term UV exposure results in histological and visible changes in the skin, including: damage to the underlying connective tissue, manifested as elastosis and increases in the glycosaminoglycans and loss of collagen; dermal accumulation of elastin-staining material resulting from the degenerative changes in collagen fibers; epidermal dysplasia with cytologic atypia and loss of polarity of keratinocytes; and an inflammatory infiltrate (see, e.g., Bissett et al. (1987) Photochemistry and Microbiology 46:367-378). The degradation of elastic fibers and wrinkling associated with intrinsically aging skin also accompanies photoaging. In humans with advanced photodamage can be detected in the changes in the staining properties of dermal tissue resulting from changes in the insoluble and soluble fractions of collagen that occur as the entire upper dermis becomes filled with elastosis (Kligman et al. (1989) J. Investigative Dermatol. 93:210-214). The changes in collagen and elastic fiber over decades of such exposure result in skin that is wrinkled, yellowed, blotchy, lax, rough and leathery. Scanning electron microscopy of aged skin shows a more dense network of elastic fibers in a more disorganized arrangement than younger skin.
Exposure to sunlight is such a pronounced factor in premature aging that by middle age individuals who have been exposed to more sunlight appear older than those who have not. The extent of dermal degenerative change correlates with the visible signs of premature aging. The subepidermal band of normal dermis, which is a site of continual dermal repair, contains normal collagen fibers. This zone becomes visually evident, however, only after there is sufficient elastotic damage to delineate this region. The elastotic material is composed principally of elastin and microfibrillar proteins that codistribute with fibronectin (see, Schwartz (1988) J. Invest. Dermatol. 91:158-161). Actinic elastosis appears to be reversible to some extent by treatment with chemical peels, dermabrasion or topical application of tretinoin (see, e.g., Warren et al. (1991) J. American Acad. Dermatol. 25:751-760; and Weiss et al. (1988) J. American Medical Assoc. 259:527-532).
The actinic mouse as a model for aging human skin
The hairless mouse is a recognized model for studying aging human skin, including studying the effects of UV radiation on the aging process and on the development of pathologies of the skin (see, e.g., Kligman et al. (1985) in Models in Dermatology, Maibach et al. (eds.), Basel, Karger, pp. 59-68; Kligman et al. (1982) J. Invest. Dermatol. 78:181-189). UV-induced connective tissue changes is similar in humans and the hairless mouse. The action spectrum and time-course for UV-induced erythema in the hairless mouse and the human is similar to the sunburn response in man (Cole et al. (1983) Photochem. Photobiol. 37:623-631).
Chronic exposure of hairless mice to UV light in amounts sufficient to cause dermal elastosis and damage leads to wrinkles similar to wrinkles seen in human skin as a result of photoaging. In the hairless mouse, the wrinkles appear as regularly spaced furrows on the dorsal surface that do not disappear upon gentle stretching. The wrinkles form when the underlying muscle contracts and the skin adapts by forming folds pedicular to the line of the contraction (Wright et al. (1973) J. Soc. Cosmet. Chem. 24:81-85). These are comparable to so-called permanent deep wrinkles that appear on sun-exposed human skin that age more rapidly than areas of the body that are not exposed.
Studies using hairless mice demonstrate that the visible appearance of the skin changes with UV exposure and that the changes mirror those seen in human skin after long-term UV exposure. As with human skin, the changes, which are manifested as wrinkling, sagging and tumor development, are a function of the quantity and quality of UV exposure. Studies using these mice have shown that skin wrinkling is induced by UVB and UVB plus UVA (315-400 nm); skin sagging is induced by high doses of UVA; tumors develop upon exposure to UVB and UVB plus UVA irradiation (see, e.g., Bissett et al. (1989) Photochem. Photobiol. 50:763-769). Exposure of the mice to UVB +UVA exposure results in thickened epidermis and changes in several components of the dermis, including thickening and proliferation throughout the upper dermis of the elastic fibers and hyperproliferation of fibroblasts and sebaceous cells and the formation of dermal cysts from remnants of follicular epithelium. The action spectrum for thickening and glycosaminoglycan increase also appears to be in the UVB range of between 285-300 nm. Ultimately, after long-term exposure (about 16 weeks), the skin becomes elastotic with thick, tangled masses of elastic fibers in the dermis. The entire dermis exhibits an increase in cellularity and an infiltrate of inflammatory cells, primarily macrophages and a few neutrophils (Bissett et al. (1987) Photochemistry and Photobiology 46:367-378).
The hairless mouse also serves as a model for testing potential treatments that reverse or repair UV-induced damage and changes associated with aging per se. Studies, using hairless mouse models, indicate that upon cessation of the UV irradiation, a band of new dermal tissue is produced in the immediate subepidermal region, which compresses the old elastotic tissue. The width of this band serves as a measure of the repair. Retinoid treatment causes dose-dependent increments in the area of the dermal repair zone. For example, topical application of all-trans retinoic acid for 10 weeks stimulates the deposition of a 100 .mu. deep subepidermal band of collagen in photoaged hairless mice (Schwartz et al. (1991) J. Invest. Dermatol 96:975-978). The resulting reconstructed dermis is thickened, contains new collagen, and the tangled, disorganized elastin, produced upon exposure to the UV irradiation, is packed into a thin layer in the lower dermis, thereby eliminating the "permanent wrinkle" on the surface.
Cytokinins
There have been a few reports suggesting the use of kinetin, a cytokinin, to promote cell division of mammalian cells and on this basis suggest its use in cosmetics. Such reports describe the use of kinetin in conjunction with other cell growth-or cell division-promoting compounds and postulate activity based on the cell division-promoting activity of kinetin. For example, French Patent 1,587,633 (27 Mar. 1970) describes a method for preparing a plant extract that is enriched in kinetin. This French Patent suggests that, because the rate of cell renewal diminishes with age, the plant extract may be used in a cosmetic formulation for the treatment of sagging skin and wrinkles by increasing the rate of cell proliferation. Japanese patent Application Publication No. 60-19709 (1985) describes a composition that contains no more than 1% of kinetin and no more than 20% of an uncharacterized lithosperum root extract and is said to accelerate cell division in human skin and thereby prevent skin-aging.
The cytokinins are a class of plant hormones defined by their ability to promote cell division in plant tissue explants in the presence of an auxin, such as indoleacetic acid, and nutrients, including vitamins, mineral salts, and sugar. In promoting cell division of plant cells, cytokinins are active at low concentrations (as low 0.01 parts per million (ppm)), but exhibit activity only in the presence of an auxin. Certain cytokinins, including zeatin (6-(3-hydroxymethyl-3-methylallyl)-aminopurine) and 6-(3,3-dimethylallyl)-aminopurine, also occur as the base moiety components of transfer RNA in yeast, bacterial, animal cells and plant cells. The cytokinin kinetin (6-furfuryl-aminopurine) forms complexes with certain RNA-binding proteins of wheat embryo extracts and appears to promote protein synthesis in plants (see, e.g,, Spirin and Ajtkhozhin (1985) Trends in Biochem. Sci., p. 162). Kinetin and other cytokinins are used in conjunction with auxin used in horticulture and in plant tissue culture, such as in the production of plantlets from plant callus tissue. Cytokinins are also used in the production of protein-rich yeast (see e.g., East German Patent No. 148,889 (1981) (Derwent World Patent Index Abstract)) and to augment the growth of microbial cultures (Merck Index, 10th Ed. (1983) Entry 5148, Merck and Co., Rahway, N. J., U.S.A.).
Certain cytokinins have been shown to inhibit the growth of tumor cells in vitro (see, e.g., Katsaros et al. (1987) FEBS Lttrs. 223:97-103). It appears that this effect is mediated via the cytotoxic affects of adenosine analogs, such as the 6-(substituted amino) purine cytokinins, that interfere with tRNA methylating enzymes (Wainfan et al. (1973) Biochem. Pharmacol. 22:493-500). When immortalized fibroblast cells are contacted with adenosine analogs the cultured cells exhibit decreased growth rate and a change in morphology from the normal flattened elongated morphology typical of cultured fibroblasts to a very elongated spindle-shape characteristic of a cytotoxic response. The very elongated shape of immortalized cells exhibiting this response is not shape characteristic of young, healthy, primary cultures of normal diploid fibroblasts.
Reversing, slowing or ameliorating the adverse effects of cellular aging
Preventing, reversing or slowing the process of cellular aging has been a persistent, though elusive, goal of biological science, that would have a number of significant and practical consequences. Preventing the aging of cells in human skin or other organs would be associated with preservation of structural and functional integrity and also cosmetic integrity. If cultured cells could be treated so that they retain characteristics of young cells, the production of valuable products by such cells in culture could be improved.
Any composition intended for use to reverse, slow, or otherwise ameliorate the adverse effects of aging that is used to treat cells in vivo or in culture that acts by increasing growth rate or total proliferative capacity of cells, however, must be viewed with caution. Since tissues, including the skin and intestinal wall, contain cells normally under tight proliferative control, any treatment to ameliorate the adverse effects of aging on cells by increasing the rate of cell division or total proliferative capacity of the cells has the potential to promote undesirable transformation of the treated cells. Thus, it is undesirable for cosmetics or pharmaceuticals intended for the treatment of humans to promote cell division, since promoting increased cell division may result in a loss of the tight proliferative control and lead to pre-cancerous or cancerous transformation of the treated cells. In addition, promoting cell division may also lead to cosmetically unacceptable changes in treated cells and tissues, such as thickening of the treated tissues.
Compositions and methods that ameliorate the adverse effects of aging on morphological, physiological, and biochemical characteristics of cells but that do not lead to potentially harmful increases in the growth rate or total proliferative capacity of the treated cells would be advantageous. Such compositions or methods are not, however, available. Also, because of the important role of protein synthesis by fibroblasts in maintaining the health of skin and other surface tissues, and because available evidence indicates that protein biosynthetic activity of fibroblasts decreases significantly with age, methods and compositions that alter the biosynthetic activity of fibroblasts in vivo, without affecting the total proliferative capacity of growth rate of such fibroblasts, would be especially advantageous.
Therefore, it is an object herein to provide compositions and methods for ameliorating the adverse effects of aging on mammalian cells in vitro and in vivo without substantially altering the growth rate or total proliferative capacity of the treated cells. In particular, it is an object herein to provide compositions and methods for reducing or reversing the wrinkling and sagging and other characteristics associated with aging human skin. It is also an object herein to provide compositions and methods for ameliorating the adverse effects of aging on cultured cells.